Adenosine A1 receptor stimulation reduces D1

Experimental Neurology 261 (2014) 733–743

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Adenosine A1 receptor stimulation reduces D1 receptor-mediated GABAergic transmission from striato-nigral terminals and attenuates L-DOPA-induced dyskinesia in dopamine-denervated mice Dalila Mango a,b,1, Alessandra Bonito-Oliva c,1, Ada Ledonne a,b, Loredana Cappellacci d, Riccardo Petrelli d, Robert Nisticò a,e, Nicola Berretta a, Gilberto Fisone c, Nicola Biagio Mercuri a,b,⁎ a

IRCCS Fondazione Santa Lucia, Rome, Italy Department of Systems Medicine, University of Rome Tor Vergata, Rome, Italy c Karolinska Institutet, Department of Neuroscience, Stockholm, Sweden d School of Pharmacy, Medicinal Chemistry Unit, University of Camerino, Italy e Department of Physiology and Pharmacology, Sapienza University of Rome, Rome, Italy b

a r t i c l e

i n f o

Article history: Received 23 May 2014 Revised 30 July 2014 Accepted 2 August 2014 Available online 27 August 2014 Keywords: Dopamine Adenosine Substantia nigra pars reticulata Levodopa Dyskinesia Mice GABAergic terminals

a b s t r a c t γ-Aminobutyric acid A receptor (GABAAR)-mediated postsynaptic currents were recorded in brain slices from substantia nigra pars reticulate neurons. The selective adenosine A1 receptor (A1R) antagonist, 8-cyclopentyl1,3-dipropylxanthine (DPCPX), increased the frequency, but not the amplitude of spontaneous inhibitory postsynaptic currents (IPSCs) in the presence of the dopamine D1 receptor agonist SKF 38393 (SKF) and phosphodiesterase 10A inhibitors (papaverine or AE90074). Under these conditions, DPCPX also increased the amplitude of evoked IPSCs (eIPSCs). The effect of DPCPX was also examined in a mouse model of Parkinson's disease (PD), generated by unilateral denervation of the dopaminergic input to the striatum. In this model, SKF alone was sufficient to increase sIPSCs frequency and eIPSCs amplitude, and these effects were not potentiated by DPCPX. To confirm a depressive effect of A1Rs on the synaptic release of GABA we used the selective A1R agonist 5′-chloro-5′-deoxyN6-(±)-(endo-norborn-2-yl)adenosine (5′Cl5′d-(±)-ENBA) which has limited peripheral actions. We found that 5′Cl5′d-(±)-ENBA decreased sIPSCs frequency, without affecting their amplitude, and decreased eIPSCs amplitude. Importantly, in the PD mouse model, 5′Cl5′d-(±)-ENBA prevented the increase in sIPSC frequency and eIPSC amplitude produced by SKF. Since exaggerated DA transmission along the striato-nigral pathway is involved in the motor complications (e.g. dyskinesia) caused by prolonged and intermittent administration of LDOPA, we examined the effect of A1R activation in mice with unilateral DA denervation. We found that 5′Cl5′ d-(±)-ENBA, administered in combination with L-DOPA, reduced the development of abnormal involuntary movements. These results indicate the potential benefit of A1R agonists for the treatment of L-DOPA-induced dyskinesia and hyperkinetic disorders providing a mechanistic framework for the study of the interaction between DA and adenosine in the striatonigral system. © 2014 Elsevier Inc. All rights reserved.

Introduction The substantia nigra pars reticulata (SNpr) is an important relay network that connects the striatum to the thalamus, the superior Abbreviations: ACSF, artificial cerebrospinal fluid; cAMP, cyclic adenosine monophosphate; DA, dopamine; DARPP-32, DA- and cAMP regulated phosphoprotein of 32 kDa; D1R, D1 receptor; A1R, A1 receptor; eIPSCs, evoked IPSCs; sIPSCs, spontaneous IPSCs; IEI, inter-event interval; fsk, forskolin; MSNs, medium-spiny neurons; PDE, phosphodiesterase; PKA, cAMP-dependent protein kinase; PP, paired pulse; papav, papaverine; SKF, SKF 38393; SNpr, substantia nigra pars reticulata. ⁎ Corresponding author at: IRCCS Fondazione Santa Lucia, via del Fosso di Fiorano, 64, 00143 Rome, Italy. E-mail address: [email protected] (N.B. Mercuri). 1 Shared co-first authorship.

http://dx.doi.org/10.1016/j.expneurol.2014.08.022 0014-4886/© 2014 Elsevier Inc. All rights reserved.

colliculus (Di Chiara et al., 1979; Yasui et al., 1995) and the brain stem (Smith et al., 1998; Takakusaki, 2013). A key regulatory role on the activity of SNpr neurons is played by the γ-aminobutyric acid (GABA)ergic afferents of striatal medium spiny neurons (MSNs). Thus, the inhibition exerted by MSNs on SNpr GABAergic neurons promotes the activity of glutamatergic thalamic neurons, maintaining an excitatory synaptic drive to the cerebral cortex (Beckstead and Frankfurter, 1982; Bodor et al., 2008; Chuhma et al., 2011; Deniau et al., 1978; Herkenham, 1979; Nishimura et al., 1997). Interestingly, the release of GABA in the SNpr is modulated by many receptors located on MSNs (Bergevin et al., 2002; Grillner et al., 2000; Lu and Ordway, 1997; Szabo et al., 2002; Wu et al., 1995; Zheng et al., 2002), critically involved in functional and dysfunctional aspects of basal ganglia transmission through modification of SNpr neurons’ discharge.

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D. Mango et al. / Experimental Neurology 261 (2014) 733–743

In Parkinson's disease (PD), the loss of the dopamine (DA) input to the basal ganglia reduces the response of D1 receptors (D1Rs) expressed on the MSNs of the striatonigral pathway, thereby contributing to the emergence of akinesia (De Long, 1990). On the other end, DA depletion produces a sensitization of D1R transmission (Gerfen et al., 2002), which, in the presence of dopaminergic drugs, may lead to an excessive release of GABA on SNpr cells. This, in turn, results in thalamic disinhibition and cortical over excitation, ultimately involved in motor complications associated with prolonged administration of L-DOPA, such as dyskinesia (Cenci et al., 2009; Feyder et al., 2011; Santini et al., 2008). We have recently demonstrated that the stimulation of D1Rs increases GABA-mediated synaptic events in SNpr cells, only in association with permissive factors such as: phosphodiesterase (PDE) 10A inhibition and presynaptic depolarization. On this regard it should be noted that there are conflicting reports on D1-mediated facilitation (Cameron and Williams, 1993; Chuhma et al., 2011; de Jesús Aceves et al., 2011; Radnikow and Misgeld, 1998) and inhibition (Miyazaki and Lacey, 1998) of IPSCs. Several factors may explain the lack of effect by the sole stimulation of D1Rs that we observed, including our animal model (mice vs. rats), the metabolic state of the GABAergic due to temperature settings, level of extracellular K+, the use of a potassium- vs. a cesiumbased intracellular solution, or agonist and antagonist concentrations. Anatomical data show that PDE10A is present in the direct D1Rbearing (striato-substantia nigra and striato-internal pallidus) and indirect D2R-bearing (striato-external pallidus) MSNs, but not in striatal interneurons (Coskran et al., 2006; Nishi et al., 2008; Sano et al., 2008; Xie et al., 2006). We have also reported that, following DA denervation, D1R stimulation alone increases synaptic GABAergic events in the SNpr (Mango et al., 2014). This phenomenon is most likely caused by the development of sensitized D1R-transmission on nigrostriatal MSNs and may be implicated in L-DOPA-induced dyskinesia. A large amount of data indicates that adenosine antagonizes D1Rmediated transmission via stimulation of A1 receptors (A1Rs) (Ferré, 1997; Ferré et al., 2001), which are densely expressed in the SNpr (Fastbom et al., 1987). It has been shown that D1Rs and A1Rs are co-localized in striatum (Ferré et al, 1991); thus, an opposing interaction between A1 and D1 receptors may take place at the level of second messengers and beyond (Abbracchio et al, 1987; Fuxe et al, 2007). Thus, by using patch-clamp electrophysiological techniques, we examined the effects of the A1R antagonist 8-cyclopentyl-1,3dipropylxanthine (DPCPX) and the potent and highly selective A1R agonist 5′-chloro-5′-deoxy-N6-(±)-(endo-norborn-2-yl) adenosine (5′-chloro-5′-deoxy-(±)-ENBA, 5′Cl5′d-(±)-ENBA) which has limited peripheral actions (Franchetti et al., 2009; Luongo et al., 2012, 2014) on the GABAergic synaptic transmission in the normal and DA-depleted SNpr. Moreover, we studied the impact of the A1R–D1R interaction in vivo, using a mouse model of PD and L-DOPA-induced dyskinesia. Materials and methods Animals Male C57BL/6 mice (Taconic, Tornbjerg, Denmark) were housed under a 12-hour light-dark cycle with food and water ad libitum. Behavioral experiments were carried out during the light phase. All experiments followed international, as well as local guidelines on the ethical use of animals from the European Communities Council Directive of 24 November 1986 (86/609/EEC), the ethical committee of the University of Tor Vergata (Rome, Italy) and the Swedish Animal Welfare Agency. All efforts were made to minimize animal suffering and the number of animals used. Slice preparation C57BL/6 mice (about 30 days old) were anesthetized with intraperitoneal (i.p.) injection of chloral hydrate (400 mg/kg) and killed by

decapitation. Slices containing both the ventral midbrain and the striatum were cut as previously described (Mango et al., 2014). A single slice was then placed in a recording chamber (~0.5 ml volume), on the stage of an upright microscope (Axioscope FS, Carl Zeiss, Germany) and submerged in a continuously flowing (3 ml/min) ACSF at 34–35 °C, composed of (in mM): NaCl 126; KCl 2.5; MgCl2 1.2; CaCl2 2.4; NaH2PO4 1.2; NaHCO3 19; glucose 11; and saturated with 95% O2 and 5% CO2 (pH 7.4). Drugs The following pharmacological agents were used (from SigmaAldrich, Milan, Italy): (5R,10S)-(−)-5-methyl-10,11-dihydro-5Hdibenzo[a,d]cylcohepten-5,10-imine maleate (MK-801), 6-cyano-7nitroquinoxaline-2,3-dione disodium salt hydrate (CNQX), papaverine, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), SCH 23390, (±)-1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol hydrobromide (SKF 38393), 6-chloro-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine7,8-diol (SKF 81297), 6-hydroxydopamine hydrochloride (6-OHDA), and 3,4-dihydroxy-L-phenylalanine (L-DOPA) (administered i.p. together with benserazide hydrochloride). AE90074 was a kind gift of Dr Jan Kehler, Lundbeck (Copenhagen, Denmark) and N6-(±)-endo-norbornyl9H-(5-chloro-5-deoxy-β-D-ribofuranosyl)adenine (5′-chloro-5′-deoxy(±)-ENBA, 5′Cl5′d-(±)-ENBA) was synthesized at the University of Camerino, as previously reported (Franchetti et al., 2009). All drugs used in the electrophysiological experiments were bath applied, while those for the behavioral experiments were dissolved in saline and injected i.p. in a volume of 10 ml/kg, except for 5′Cl5′d-(±)-ENBA that was dissolved in 5% DMSO. Electrophysiology Whole cell patch-clamp recordings were obtained as previously described (Mango et al., 2014) using borosilicate glass electrodes (3–5 MΩ) filled with (in mM): KCl (145), CaCl2 (0.05), EGTA (0.1), HEPES (10), Na3-GTP (0.3), and Mg-ATP (4.0) (pH adjusted to 7.3 with KOH). Spontaneous IPSCs (sIPSCs) were recorded in the continuous presence of MK-801 (10 μM) and CNQX (10 μM), in order to block ionotropic glutamate receptors, and captured off-line from 3 min traces using Clampfit (Molecular Devices, Sunnyvale, CA, USA). Stimulusevoked IPSCs (eIPSCs) were generated with a bipolar stimulating electrode in the striatum. A paired-pulse protocol (PPR) was employed with an inter-pulse interval of 50 ms to evaluate changes in the pairedpulse ratio (PPR) of eIPSCs (PPR = 2nd IPSC/1st IPSC). 6-OHDA lesions Twenty-one day (young) or 2 month (adult) old mice were anesthetized with a mixture of Hypnorm Solution (VetaPharma Ltd, Leed, UK), Midazolam 5 mg/ml (Hameln Pharmaceuticals GmbH, Hameln, Germany) and water (1:1:2) and mounted in a stereotaxic frame (David Kopf Instruments, Tujunga, CA, USA) equipped with a mouse adaptor. 6-OHDA was dissolved in saline containing 0.02% ascorbic acid at the concentration of 3 μg/μl free base. Each mouse received two unilateral injections (2 μl each) of 6-OHDA into the right striatum as previously described (Santini et al., 2007), according to the following coordinates (mm) (Franklin and Paxinos, 1997): for young mice — anteroposterior (AP) +1, mediolateral (ML) −1.9, dorsoventral (DV) −2.9 and AP +0.5, ML −2.2, DV −2.9; for adult mice — AP +1, ML − 2.1, DV − 3.2 and AP + 0.3, ML − 2.3, DV − 3.2. This procedure leads to an ≥80% decrease in striatal tyrosine hydroxylase (TH) immunoreactivity (see Fig. 5A), as assessed post-mortem by Western blotting in all mice. Only the animals with this level of depletion were included in the analysis. One week after the lesion, young mice were subject to an electrophysiological investigation. In preliminary experiments, a group of young mice prepared according to the same procedure was also


examined for TH depletion, akinesia, and responsiveness to D1R activation, in order to confirm their validity as a PD model. Two-month old mice were allowed to recover for 3 weeks and then tested for motor activity or L-DOPA-induced dyskinesia. Western blotting Mice unilaterally lesioned with 6-OHDA were sacrificed by decapitation. The left and right striata were dissected out, sonicated in 750 μl of 1% SDS and boiled for 10 min. Brain samples were processed as previously described (Santini et al., 2007) and loaded (5 μg) onto 10% SDS–polyacrylamide gel. Proteins were separated by electrophoresis and transferred overnight to polyvinylidene difluoride membranes (Amersham Pharmacia Biotech, Uppsala, Sweden) (Towbin et al., 1979). The membranes were immunoblotted using antibodies against total GluA1, GluA1 phosphorylated at Ser845 (PhosphoSolution, 1:10,000 and 1:1000, respectively) and TH (1:3000, Chemicon International, Massachusetts), and then incubated in horseradish peroxidase-conjugated secondary anti-mouse antibody (1:30,000). The signal was visualized by ECL (Pierce, Rockford, IL) and quantified using Quantity One software (Bio-Rad). The levels of phosphor-Ser845-GluA1 were normalized for the amount of total GluA1 detected in the sample. The unlesioned striatum was used as control, as in the previous studies (Santini et al., 2007). Behavioral analyses Horizontal motor activity was measured in adult mice using the novel home cage test. The animals were injected with saline or 0.5 mg/kg 5′Cl5′d-(±)-ENBA and individually placed in a novel home cage (Bonito-Oliva et al., 2013). Motor activity was video-recorded for 1 h, and the distance (cm) covered by each animal was analyzed with Biobserve GmbH software (St. Augustin, Germany). The cylinder test (Schallert and Tillerson, 2000) was used to assess akinesia in young and adult mice, 1 week and 3 weeks after the 6-OHDA lesion, respectively. The animals were treated with saline or L-DOPA (young mice), or with saline, L-DOPA or L-DOPA plus 5′Cl5′d(±)-ENBA (adult mice). One hour after drug administration, the mice were individually placed in transparent glass cylinders, and the wall contacts performed with extended digits were video-recorded for 10 min (Lundblad et al., 2004). The number of contacts made with the right and left forelimbs was counted and compared by an observer blind to the experimental group to monitor akinesia (expressed as reduced number of wall contacts with the forelimb contralateral to the side of the lesion). Abnormal involuntary movements (AIMs) were assessed in adult 6-OHDA-lesioned mice, after 9 days of L-DOPA, or L-DOPA plus 5′Cl5′ d-(±)-ENBA administration, using a previously established protocol (Santini et al., 2007). Twenty minutes after the last drug injection, mice were placed in individual cages and dyskinetic behavior was assessed for 1 min every 20 min, over a period of 2 h. AIMs were classified into four subtypes: locomotive (contralateral turn), axial AIMs (dystonic posturing of the upper part of the body toward the side contralateral to the lesion), limb AIMs (abnormal movements of the forelimb contralateral to the lesion), and orolingual AIMs (vacuous jaw movements and tongue protrusion). Each subtype was scored on a severity scale from 0 to 4 (absent to continuous and not interruptible). Axial, limb and orolingual AIMs were individually scored and the data were then pulled together and expressed as ALO. Statistics Drug effects on sIPSCs were evaluated as changes in their amplitude or inter-event interval (IEI) using the Kolmogorov–Smirnov (K–S) test on their mean cumulative distributions (bin size of 50 ms for IEI and 10 pA for amplitude), or by comparison of their mean median

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values with the Student's t-test (t-test). Changes in eIPSCs amplitude (mean ± SEM) in response to specific pharmacological treatments were compared using the Student's t-test. Behavioral data were analyzed by one-way ANOVA (treatment), followed by Fisher's post-hoc comparison, for the cylinder test, or two-way ANOVA (treatment and time) with repeated measure (time) for the AIMs time-course. In all other experiments, t-test was used for comparisons between the two groups. A p b 0.05 was considered significant and n means the number of neurons or animals. Results All electrophysiological data were obtained from neurons characterized by a tonic firing rate (N 5 Hz), with no or small voltage sag when negative current steps were applied in current clamp-mode, or, with hyperpolarization-activated, time-dependent inward current (Ih) not as large as that typically found in DA neurons, when recorded in voltage-clamp mode. These properties have been indicated for the electrophysiological identification of GABAergic SNpr neurons (Berretta et al., 2000; Ibañez-Sandoval et al., 2006; Lee and Tepper, 2007; Lee et al., 2011; Nakanishi et al., 1987; Shen and Johnson, 1997; Stanford and Lacey, 1996). SNpr neurons receive extensive GABAergic inputs from both the striatum and the globus pallidus (Ribak et al., 1980; Tepper and Lee, 2007), thus undermining the possibility to discriminate the striatal input within the whole sIPSCs population. However, the use of parasagittal slices containing both the ventral midbrain and the striatum, allowed us to match our analysis on sIPSCs with the response of eIPSCs obtained by placing the stimulating electrode in the striatum of striato-nigral slices, purposely cut in order to partially preserve the striato-nigral direct pathway (Connelly et al., 2010; Mango et al., 2014). The selective recruitment of the striato-nigral projection was further confirmed by the presence of eIPSCs paired-pulse facilitation (PPF) in response to the pair of stimuli (50 ms apart; see Fig. 3B). This form of short-term plasticity has been shown to discriminate striato-nigral IPSCs from those originating from globus pallidus-nigral terminals (Connelly et al., 2010; de Jesús Aceves et al., 2011). All recordings were obtained in the presence of MK-801 (10 μM) and CNQX (10 μM), in order to block ionotropic glutamate receptors. An endogenous adenosine tone regulates the spontaneous release of GABA mediated by D1Rs The interplay between A1Rs and D1Rs in the SNpr was investigated by examining the effect exerted by tonic and endogenous A1R stimulation on the D1R-mediated enhancement of the GABAergic transmission. sIPSCs amplitude and IEI were not affected by the A1R antagonist DPCPX (200 nM) perfused alone (p N 0.05, K–S test and t-test, ctrl vs. DPCPX, n = 12; Fig. 1A), or in combination with the D1R agonist SKF (10 μM) (p N 0.05 for both sIPSCs amplitude and IEI, K–S test and t-test, DPCPX vs. DPCPX + SKF, n = 10; Fig. 1A). However, we evaluated the effect of DPCPX + SKF on GABAergic transmission, in the presence of the PDE10A inhibitors. As shown in Fig. 1B, we did not observe any effect on the sIPSCs amplitude (p N 0.05, K–S test and t-test; Fig. 1B left), but sIPSCs IEI was reduced when DPCPX and SKF were applied in the presence of papaverine (50 nM) (p b 0.001, K–S test, compared to DPCPX + SKF alone; n = 7; Fig. 1B right), or AE90074 (50 nM) (p b 0.01, K–S test, compared to DPCPX + SKF alone; n = 7; Fig. 1B right). These observations indicate the presence of an antagonistic interaction between D1Rs and A1Rs in the regulation of GABAergic transmission in the SNpr. Tonic A1R stimulation and D1R-mediated facilitation of evoked IPSCs The existence of a negative coupling between A1Rs and D1Rs was also verified on striatonigral eIPSCs. The A1R antagonist DPCPX

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Fig. 1. Endogenous adenosine tone regulates the D1R-mediated effects on GABA release. A) Pooled cumulative distributions of sIPSCs amplitude (left; bin size 10 pA) and inter-event interval (IEI) (right; bin size 50 ms) recorded from neurons in response to DPCPX alone (n = 12) or DPCPX plus SKF 10 μM (n = 10). Histograms in the insets are averages (mean ± S.E.M.) of the corresponding median values. On top are representative traces. (Note the lack of effects in both conditions.) B) Pooled cumulative distributions of sIPSCs amplitude (left) and IEI (right) recorded from neurons in response to DPCPX + SKF and co-application of papaverine (papav) (n = 7) or AE90074 (n = 7). Histograms in the insets are averages (mean ± S.E.M.) of the corresponding median values. On top are representative traces. C) Histograms (mean ± S.E.M.) of the eIPSCs amplitude recorded from neurons in response to DPCPX + SKF (n = 12) and co-application of papav (n = 6) or AE90074 (n = 6) and SCH 23390 (n = 12). The representative traces, shown above, are obtained from the same neurons treated with papav (a) or AE90074 (b). DPCPX + SKF (left), during papav (a, center) or AE90074 (b, center) and SCH 23390 application (right). *p b 0.05 t-test. D) Histograms (mean ± S.E.M.) indicate changes of the eIPSCs amplitude induced by papav in two conditions (with or without DPCPX).


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(200 nM), either alone or in the presence of the D1R agonist SKF (10 μM) failed to affect eIPSC amplitude (p N 0.05, t-test for both DPCPX alone, n = 6, and DPCPX + SKF, n = 10; Fig. 1C). However, when papaverine (50 nM) or AE90074 (50 nM) was added to DPCPX and SKF, a significant increase of the eIPSC amplitude was observed (p b 0.05, t-test, DPCPX + SKF vs. DPCPX + SKF + papaverine, n = 6; p b 0.01, t-test, DPCPX + SKF vs. DPCPX + SKF + AE90074, n = 6; Fig. 1C). This effect was blocked by the D1R antagonist SCH 23390 (1 μM) (Fig. 1C). Importantly, in the presence of papaverine, the increase in eIPSC amplitude produced by SKF was enhanced by DPCPX (p b 0.05, t-test, SKF vs. DPCPX; Fig. 1D).

DPCPX + SKF, n = 6; Fig. 2A right), without changing their amplitude (p N 0.05, K–S test and t-test, DPCPX vs. DPCPX + SKF, n = 6; Fig. 2A left). As in the case of sIPSCs, eIPSCs in DA-denervated mice were insensitive to DPCPX (p N 0.05, t-test, n = 6), while their amplitude was increased by the subsequent addition of SKF (10 μM) (p b 0.05, t-test, ctrl vs. SKF, n = 6; Fig. 2B). Of note, both the increases in sIPSCs frequency and eIPSCs amplitude induced by DPCPX plus SKF were not significantly higher than those attained in the presence of SKF alone in the DA-denervated model (p N 0.05, t-test, SKF vs. DPCPX; Figs. 2C, D). These data suggest a negligible contribution of endogenous adenosine to control the release of GABA in the DA-denervated SNpr.

Validation of 6-OHDA-induced PD model in young mice

In vitro and in vivo effects of the A1R agonist 5′Cl5′d-(±)-ENBA

The validity of unilateral 6-OHDA injections in the dorsal striatum to model PD has been previously assessed (Cenci and Lundblad, 2007; Lundblad et al., 2004, 2005). In this work, due to technical limitations of patch-clamp recordings of small size SNpr neurons, the electrophysiological experiments were performed in younger (30 days) mice, examined 1 week after 6-OHDA lesion. In a series of preliminary experiments we determined whether, also in this case, 6-OHDA was able to provide a model of PD, as in adult (≥2 months) animals. One week after surgery, sham- and 6-OHDA-lesioned mice (30 days of age) were examined in the cylinder test to estimate the degree of motor impairment (akinesia) produced by the DA denervation. We observed that the lesion caused a strong reduction in the use of the left forepaw (controlateral to the lesion) (22.1% ± 4.2 of total wall contacts) as compared to sham mice (52.1% ± 1.8 of total wall contacts). One-way ANOVA indicates a significant effect of the lesion on the use of the left forepaw (F(1,15) = 42.9, p b 0.0001, n = 8/group). The motor deficit observed in the lesioned mice was reversed by administration of L-DOPA (6 mg/kg, plus 4.5 mg/kg benserazide) (50.9% ± 4.4 of total wall contacts for the forepaw controlateral to the side of the 6-OHDA lesion). At the end of this experiment, the animals were sacrificed and tyrosine hydroxylase was quantified by Western blotting to assess DA depletion. The analysis showed a significant reduction in the 6-OHDA-injected striatum as compared to the unlesioned striatum [p b 0.0001, Student's t-test]. We also evaluated the ability of the D1R agonist, SKF 81297 (3 mg/kg) to increase the cAMP-dependent phosphorylation of GluA1 in the striatum of 6-OHDA-lesioned young mice. Western blot analysis indicated a sensitized response in the 6-OHDA-lesioned striatum compared to the unlesioned striatum. Two-way ANOVA revealed a significant interaction between lesion and treatment [F(3,15) = 9.66, p b 0.05]. Post-hoc comparisons indicated that the increase in phosphorylation of GluA1 at Ser845 produced by SKF 81297 in the lesioned striatum (692.3 ± 360% of control) was significantly higher than that produced in the unlesioned striatum (294.8 ± 74.6% of control). Altogether, these results indicate that, in 30-day old mice, the effects observed 1 week after injection of 6-OHDA in the striatum are comparable to those observed in adult mice.

It is known that adenosine, acting on A1Rs, reduces GABA release in the SNpr (Shen and Johnson, 1997). In line with these data, we found that, in slices of SNpr from control mice, the selective A1R agonist 5′Cl5′d-(±)-ENBA (1 μM) slightly increased sIPSCs IEI (p b 0.05, K–S test, ctrl vs. 5′Cl5′d-(±)-ENBA n = 8; Fig. 3A right) without changing their amplitude (p N 0.05, K–S test, ctrl vs. 5′Cl5′d-(±)-ENBA n = 8; Fig. 3A left). We also recorded eIPSCs and found that the A1R agonist 5′Cl5′d-(±)ENBA reduced their amplitude (p b 0.001, t-test, n = 6) with a parallel increase of PPR (p b 0.05, t-test, n = 6, Fig. 3B), indicating a presynaptic mechanism of action. To evaluate the effects produced by the A1R stimulation on motor function we examined locomotion in mice treated with saline or 0.5 mg/kg of 5′Cl5′d-(±)-ENBA, using the novel home cage test. As shown in Fig. 3C, 5′Cl5′d-(±)-ENBA significantly reduced the horizontal locomotor activity (p b 0.01, t-test).

Endogenous A1R stimulation and D1R-mediated facilitation of the GABAergic transmission in DA-denervated mice It has previously been reported that DA depletion potentiates the effects of D1R agonists on nigrostriatal MSNs (Gerfen et al., 2002). Such a potentiation is reflected by the ability of SKF to increase GABAergic transmission without concomitant inhibition of PDE10A (Mango et al., 2014). Therefore, we tested the D1R vs. A1R interaction in mice lesioned with 6-OHDA. DPCPX (200 nM) applied alone did not affect sIPSCs amplitude (p N 0.05, K–S test and t-test, ctrl vs. DPCPX, n = 6; Fig. 2A left) nor the sIPSCs IEI (p N 0.05, K–S test and t-test, ctrl vs. DPCPX, n = 6; Fig. 2A right). However, when SKF (10 μM) was co-applied, DPCPX significantly reduced sIPSCs IEI (p b 0.001, K–S test, DPCPX vs. DPCPX + SKF, n = 6; p b 0.05, t-test, DPCPX vs.

Inhibition of the GABAergic transmission by A1Rs and interaction with D1Rs in conditions of DA-denervation In the next set of experiments, we examined the effect of A1R stimulation on D1R transmission in DA-denervated mice. We observed that the decrease of the IEI induced by SKF was reduced in the presence of the specific A1R agonist 5′Cl5′d-(±)-ENBA (1 μM). In fact, 5′Cl5′d(±)-ENBA induced a significant rightward shift of the IEI cumulative probability plot (p b 0.01, K–S test, SKF vs. 5′Cl5′d-(±)-ENBA n = 7; Fig. 4A right) with no change in their amplitude (p N 0.05, K–S test, SKF vs. 5′Cl5′d-(±)-ENBA n = 7; Fig. 4A left). Moreover, after DA-denervation 5′Cl5′d-(±)-ENBA reversed the increase in the eIPSCs amplitude induced by SKF (p b 0.01, t-test, SKF vs. 5′Cl5′d-(±)-ENBA, n = 6, Fig. 4B). This effect on eIPSCs was paralleled by changes of the PPR, suggesting a presynaptic site of action by A1Rs (p b 0.05, t-test, SKF vs. 5′Cl5′d-(±)-ENBA, n = 6, Fig. 4B). 5′Cl5′d-(±)-ENBA does not affect the anti-akinetic properties of L-DOPA but reduces L-DOPA-induced dyskinesia The negative interaction between A1R- and D1R-mediated transmissions described above suggests that A1R counteracts the sensitization produced on D1Rs by DA depletion, implicated in dyskinesia. Therefore, we evaluated the effect of 5′Cl5′d-(±)-ENBA on L-DOPAinduced dyskinesia. 6-OHDA striatal injection in adult mice produced a large (≥ 80%) decrease of TH in the striatum (Fig. 5A), and three weeks later injected animals were examined in the cylinder test. In line with a previous work (Santini et al., 2007), 6-OHDA-lesioned mice treated with saline showed akinesia (expressed as reduced number of wall contacts performed with the forelimb contralateral to the side of the lesion), as compared with sham-lesioned, control mice (solid line) (Fig. 5B). L-DOPA administered alone reduced the akinesia induced by 6-OHDA (Fig. 5B). Importantly, lesioned mice that received L-DOPA in combination with 5′Cl5′d-(±)-ENBA showed a recovery of the use of the limb contralateral to the lesion similar to that observed in

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Fig. 2. Endogenous A1R stimulation and facilitation of D1R-mediated GABA release in DA-denervated mice. A) Pooled cumulative distributions of sIPSCs amplitude (left; bin size 10 pA) and inter-event interval (IEI) (right; bin size 50 ms) recorded from SNpr neurons of 6-OHDA lesioned mice, in response to DPCPX 200 nM alone (n = 6) or DPCPX plus SKF 10 μM (n = 6). Histograms in the insets are averages (mean ± S.E.M.) of the corresponding median values. The traces on top are obtained from the same neuron in control conditions, during DPCPX alone or DPCPX + SKF. B) Histograms (mean ± S.E.M.) of the eIPSCs amplitude recorded from neurons in response to DPCPX alone or DPCPX + SKF (n = 6). On the top are representative traces obtained from the same neuron, in DPCPX alone or DPCPX + SKF. *p b 0.05 t-test. C) Histograms (mean ± S.E.M) indicate changes of sIPSCs (IEI) induced by SKF in the two conditions, with or without DPCPX. D) Histograms (mean ± S.E.M.) indicate changes of eIPSCs induced by SKF in the two conditions, with or without DPCPX.


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Fig. 3. 5′Cl5′d-(±)-ENBA decreases GABA release and the motor activity in wild-type mice. A) Pooled cumulative distributions of sIPSCs amplitude (left; bin size 10 pA) and inter-event interval (IEI) (right; bin size 50 ms) recorded from SNpr neurons in response to 5′Cl5′d-(±)-ENBA 1 μM (n = 8). On top are representative traces. B) Histograms (mean ± S.E.M) of the eIPSCs amplitude (left) and paired-pulse ratio (PPR; right) recorded from neurons in response to application of 5′Cl5′d-(±)-ENBA (n = 6). On top are representative traces obtained from the same neuron, in control and 5′Cl5′d-(±)-ENBA. C) The effect of 5′Cl5′d-(±)-ENBA on motor activity was tested in 2-months old wild-type mice by the Novel Home Cage test. The animals were injected with saline (n = 8) or 5′Cl5′d-(±)-ENBA (0.5 mg/kg, n = 7) and their motor activity was measured during a 1 h test and expressed as total distance moved (in centimeters). Data are presented as mean ± SEM. *p b 0.05 t-test.

mice treated with L-DOPA alone [one-way ANOVA F(2,37) = 26.100, p b 0.0001, followed by Fisher's post-hoc comparison] (Fig. 5B). Moreover, 5′Cl5′d-(±)-ENBA did not affect the overall activity in the cylinder test, since the total wall contacts (performed with right and left forelimbs) was similar in mice treated with L-DOPA, with or without 5′Cl5′d-(±)-ENBA (106 ± 5.8 and 98 ± 2.8, respectively). These results indicate that 5′Cl5′d-(±)-ENBA does not interfere with the ability of L-DOPA to rescue motor function in a mouse model of PD. We proceeded by evaluating the ability of 5′Cl5′d-(±)-ENBA to counteract L -DOPA-induced dyskinesia. Two-month old mice were lesioned unilaterally with 6-OHDA and treated for 9 days with L-DOPA (6 mg/kg) in combination with benserazide (4.5 mg/kg), with or without 5′Cl5′d-(±)-ENBA (0.5 mg/kg). Abnormal involuntary movements (AIMs) were scored on day 9, starting 20 min after the injection. As shown in Figs. 5C–F, L-DOPA treatment induced severe AIMs, measured as locomotive (Figs. 5C, E) as well as axial, limb and orofacial (ALO) (Figs. 5D, F) involuntary movements. Interestingly, when L-DOPA was administered together with the A1R agonist 5′Cl5′d-(±)-ENBA, the animals showed a reduced severity of both locomotive and total ALO involuntary movements, as revealed by the statistical analysis (p b 0.01 for both, t-test, Figs. 5E-F).

Discussion The release of GABA from MSNs afferents to the ventral mesencephalon is regulated by various neurotransmitters acting on specific receptors (Bergevin et al., 2002; Grillner et al., 2000; Lu and Ordway, 1997; Szabo et al., 2002; Wu et al., 1995; Zheng et al., 2002). In this study, we show that endogenous adenosine acting on A1Rs blunts the ability of D1Rs to enhance the spontaneous GABAergic synaptic activity in the SNpr. The results on sIPSCs were matched by results on eIPSCs elicited on SNpr neurons in response to the direct stimulation of the striatum in combined striato-nigral slices. Indeed, the fact that we observed a facilitation of the paired-pulse evoked response is indicative that we activated the so-called “direct” basal ganglia pathway which synapses onto GABAergic projection neurons of the SNpr (Connelly et al., 2010). Interestingly, in naïve mice, the block of A1Rs by DPCPX did not affect the spontaneous and stimulus-evoked GABAergic synaptic transmission, suggesting a negligible engagement of A1Rs in normal conditions. However, when DPCPX was applied in a condition of D1R stimulation and PDE10A inhibition, it induced a facilitation of sIPSCs and eIPSCs. In this regard, it should be noted that the stimulation of adenylyl cyclase produced by activation of D1Rs, in combination with

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Fig. 4. The A1R agonist 5′Cl5′d-(±)-ENBA reduces the facilitation of the D1R-mediated GABA release in SNpr of DA-denervated mice. A) Pooled cumulative distributions of sIPSCs amplitude (left; bin size 10 pA) and inter-event interval (IEI) (right; bin size 50 ms) recorded from SNpr neurons of 6-OHDA lesioned mice, in response to SKF 10 μM alone (n = 7) or SKF plus 5′Cl5′d-(±)-ENBA 1 μM (n = 7). Histograms in the insets are averages (mean ± S.E.M.) of the corresponding median values. The traces on top are obtained from the same neuron in control conditions, during SKF alone or SKF plus 5′Cl5′d-(±)-ENBA. B) Histograms (mean ± S.E.M.) of the eIPSCs amplitude (left) and paired-pulse ratio (PPR; right) recorded from neurons in response to application of SKF 10 μM alone or SKF plus 5′Cl5′d-(±)-ENBA (n = 6). On top are representative traces obtained from the same neuron, in control, SKF alone or SKF plus 5′Cl5′d-(±)-ENBA. *p b 0.05 t-test.

PDE10A inhibition, might produce a substantial increase in cAMP, which has been proposed to represent an important source of extracellular adenosine (Barber and Butcher, 1981; Bonci and Williams, 1996; Brundege et al., 1997; Dunwiddie, 1985; Manzoni et al., 1998; Rosenberg and Ditchter, 1989; Rosenberg et al., 1994). Thus, it is possible that the combined actions of SKF and the PDE10A inhibitors papaverine/AE90074 lead to increased levels of extracellular adenosine, thus promoting the activation of A1Rs. In this circumstance, the blunting effect produced by the A1R stimulation on GABA release can be more easily unveiled by the antagonistic action of DPCPX. If this is also true in vivo, an “on demand” production of adenosine (Harvey and Lacey, 1997; Yamamoto et al., 1988) caused by an exaggerated stimulation

of D1Rs may represent a mechanism to control the excessive DAinduced release of GABA in the SNpr. However, in vitro studies performed in rat SNpr showed that endogenous adenosine, by acting on A1Rs, exerts per se a tonic inhibitory influence on GABA release (Shen and Johnson, 1997). The partial discrepancy between these previous data with the present study could be due to a different metabolic state of neurons/terminals in rats versus mice (i.e. less extracellular adenosine to occupy the A1Rs on presynaptic GABAergic terminals in mice as compared to rats). We have previously shown that DA depletion leads to a strong sensitization of D1Rs. In this condition, D1Rs acquire the ability of promoting GABAergic synaptic function per se, without the requirement

Fig. 5. 5′Cl5′d-(±)-ENBA does not affect the anti-akinetic properties of L-DOPA but reduces its dyskinetic effects.A) The dopamine depletion induced by 6-OHDA lesion in adult mice was analyzed by Western blot quantification of tyrosine hydroxylase in striatal samples. Top row shows representative autoradigrams. Data are presented as % of controls and expressed as mean ± SEM. ***p b 0.0001 t-test. B) The akinesia induced by a unilateral injection of 6-OHDA was tested with the cylinder test in 6-OHDA lesioned mice treated with saline (n = 20), L-DOPA (6 mg/kg, n = 10) or 5′Cl5′d-(±)-ENBA (0.5 mg/kg) plus L-DOPA (n = 11). The left forelimb use was determined as % of the total number of wall contacts during a 10 minutes test. Broken line indicates the percent of the left forelimb use determined in control sham-lesioned mice (48 ± 4). One-way ANOVA, followed by Fisher's post-hoc comparison. B–E) Abnormal involuntary movements (AIMs) score in 6-OHDA-lesioned mice treated for 9 days with L-DOPA (6 mg/kg, administered together with benseradide 4.5 mg/kg) (n = 22), or L-DOPA plus 5′Cl5′d-(±)-ENBA (0.5 mg/kg) (n = 25). After 8 days of treatment, mice were injected again on day 9 and AIMs were assessed for 1 min every 20 min, over a period of 120 min. AIMs were divided in locomotive (B, D) and total ALO (axial + limb + orofacial) (C, E) and shown as time course (B–C) and as total score (D, E). Data are presented as mean ± SEM. **p b 0.01 t-test.


of concomitant PDE10A inhibition or presynaptic depolarization observed in normal conditions (Mango et al., 2014). In this study, we have examined the effect of the A1R agonist 5′Cl5′d-(±)-ENBA on IPSCs

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recorded from normal and DA-depleted slices. 5′Cl5′d-(±)-ENBA is a potent and selective A1R agonist (Cappellacci et al., 2008), characterized by a prominent central effect and little cardiovascular side effects

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compared to other agonists, due to its limited action on peripheral A1Rs (Luongo et al., 2012). 5′Cl5′d-(±)-ENBA reduced the frequency of the sIPSCs in the SNpr neurons and reduced the horizontal locomotor activity. Interestingly, we found that 5′Cl5′d-(±)-ENBA counteracted the enhancing effects exerted by D1R stimulation on GABA-mediated IPSCs, in a DA-denervated condition, indicating an A1R–D1R negative interaction. Noteworthy 5′Cl5′d-(±)-ENBA has a high liposolubility, which ensures a good brain distribution upon systemic administration, with minimal peripheral effects (Luongo et al., 2012). In experimental models of PD, sensitized D1R transmission at the level of striatonigral MSNs has been proposed to participate in the development and expression of L-DOPA-induced dyskinesia (Feyder et al., 2011). Thus, pharmacological and genetic interventions that reduce D1R-mediated activation of cAMP and extracellular signalregulated protein kinase (ERK) signaling in striatal MSNs have been shown to diminish the dyskinetic behavior (Feyder et al., 2011). These observations suggest that the ability of 5′Cl5′d-(±)-ENBA to attenuate the synaptic effects produced by sensitized D1Rs in the SNpr may be paralleled by a concomitant reduction of L-DOPA-induced dyskinesia. We observed that, despite the reduction of motor activity exerted in wild-type mice, this selective A1R agonist did not affect the antiakinetic properties of L-DOPA nor the total vertical activity, in the unilaterally DA-denervated mice. More importantly, when chronically co-administered with L-DOPA, 5′Cl5′d-(±)-ENBA reduced the locomotive, as well as the axial, limb and orofacial AIMs normally produced by the antiparkinsonian drug (Cenci et al., 2009; Santini et al., 2008). Altogether, these data confirm the hypothesis that a presynaptic inhibition of GABA release achieved by A1R activation may increase SNpr neuronal activity, thus reducing dyskinesia through the inhibition of thalamic neurons (Shirakawa and Tamminga, 1994). The A1R agonist did not inhibit the increase in cAMP and ERK signaling induced by L-DOPA in the striatum (data not shown). Based on the behavioral observations, and the results obtained with the electrophysiological investigations, we propose that: 1) 5′Cl5′d-(±)-ENBA acts on A1Rs located in the terminals of striatonigral MSNs, and 2) the antidyskinetic effect of 5′Cl5′d-(±)-ENBA is mainly exerted by reducing the abnormal activation of GABAergic transmission produced by sensitized D1Rs in the SNpr. Technical limitation precluded the possibility to obtain reliable electrophysiological recordings of small size SNpr neurons in slices of mice older than 5 weeks. However, given the consistency of the electrophysiological and the behavioral data, we assume that the effects of the A1R stimulation are similar in young and adult denervated animals. Our electrophysiological findings support the notion of an antagonistic A1R–D1R interaction, likely due to the existence of A1R–D1R heteromeric complexes and opposite regulation on signal transduction (Fuxe et al., 2007). Accordingly, an antagonistic crosstalk exerted by A1Rs and D1Rs on adenylyl cyclase activity, immediate early gene expression, GABA release and motor activity has been previously documented. Therefore, the pharmacological stimulation of A1Rs has been shown to counteract the ability of D1R to increase GABA release in the strio-endopenducolar pathway (Ferré et al., 1997, 1999). Biochemical experiments evaluating Ca2+-dependent [3H]GABA release have also reported a reciprocal A1R–D1R inhibition in the SNpr (Floràn et al., 2002). Moreover, in animal models of PD, the motor effects produced by SKF are decreased by the A1R agonist, N6-cyclopentyladenosine (CPA) (Ferré et al., 1999), and potentiated by the A1R antagonist DPCPX (Floràn et al., 2002). It is interesting to note that the interaction of adenosine with dopamine in the basal ganglia is not only limited to A1 and D1 receptors, but a well described interaction exists between A2 receptors mainly located on striato-pallidal neurons and D2 receptors. It has been demonstrated that the formation of heterometric complexes between these receptors improves motor symptoms in animal models of Parkinson's disease (PD) and in initial clinical trials (for a review see Morelli et al., 2007).

Conclusions In view of the sensitivity of the GABAergic transmission in the striatonigral direct pathway to the depressant action of 5′Cl5′ d-(±)-ENBA and its effect in reducing dyskinesia, A1Rs are likely to play a critical role in the regulation of synaptic transmission in the basal ganglia, in both physiological and pathological conditions. Thus, we suggest that A1R agonists like 5′Cl5′d-(±)-ENBA that have reduced cardiovascular effects may ameliorate dyskinetic and hyperkinetic movements associated with L-DOPA treatment of PD patients, without affecting its therapeutic anti-akinetic response.

Acknowledgments This work was supported by the grant RF08.32 from Ministero della Salute, by Fondo di Ricerca di Ateneo (FAR) from the University of Camerino, Swedish Research Council, StratNeuro at Karolinska Institutet grant 13482, and Ahlèn-stiftelsen.

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